1. Field of the Invention
The invention relates to immune stimulation by chemically modified palindromic oligonucleotide analogs.
2. Summary of the Related Art
Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and interferon-gamma synthesis and enhance natural killer activity. Tokunaga et al., J. Natl. Cancer Inst. 72 (1984) 955-96. Pisetsky et al.; Reich et al., Mol. Biol. Rep. 18 (1993) 217-221; Krieg et al., Yi e al., Nature 374 (1995) 546-549 and Sato et al., Science 273 (1996) 352-354 teach that bacterial DNA, synthetic oligodeoxynucleotides, and DNA vaccines containing unmethylated CpG-dinucleotides in specific sequence contexts (CpG DNA) activate the vertebrate immune system.
Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses. TLRs recognize a wide variety of ligands, called pathogen-associated molecular patterns (PAMPs). Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defense responses through their intracellular signaling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88. Dendritic cells and macrophages normally respond to Toll-like receptor (TLR) ligands and cytokines (for example, interleukin-1β; IL-6 and tumor necrosis factor, TNF), which they also produce; natural killer (NK) cells and T cells are also involved in the pro-inflammatory circuit. After TLR stimulation by bacterial compounds, innate immune cells release a range of cytokines and chemokines. Some examples of TLR ligands include, but are not limited to, lipoproteins; peptidoglycan, zymosan (TLR2), double-stranded RNA, polyI:polyC (TLR3), lipopolysaccharide, heat shock proteins, taxol (TLR4), flagellin (TLR5), and imidazoquinolines-R848, resiquimod, imiquimod; ssRNA (TLR7/8), beta-lymphocytes (TLR10), and profilium like molecules and uropathogenic E. coli (TLR11).
Krieg et al., Annu. Rev. Immunol. 20 (2002) 709-760; Dalpke et al., Biol. Chem. 383 (2002) 1491-1500 and Kandimalla et al., Curr. Opin. Mol. Ther. 4 (2002) 122-129 teach that CpG DNAs induce innate immune cells to produce Th1 cytokines that promote cytotoxic T lymphocyte (CTL) responses and production of immunoglobulins by B cells. The immune stimulatory properties of CpG DNAs have allowed their use as therapeutic agents for a broad spectrum of disease indications including cancers, viral and bacterial infections, inflammatory disorders and as adjuvant in immunotherapy.
In addition to chemical modifications, a number of structural modifications influence the activity of CpG DNAs. Kandimalla et al., Nucleic Acids Res. 30 (2002) 4460-4469 teaches that CpG DNAs that contained two freely accessible 5′-ends through a 3′-3′-linkage had greater activity than did conventional CpG DNAs containing multiple copies of CpG motifs and a single 5′-end.
Kandimalla et al., Biochem. Biophys. Res. Commun. 306 (2003) 948-953 teaches that the presence of a secondary structure in CpG DNAs significantly affected their activity depending on the position and nature of the secondary structure, that the presence of a hairpin structure at the 5′-end abrogated stimulatory activity, and that the same structure at the 3′-end had an insignificant effect on stimulatory activity but caused lower IL-6 secretion and contributed to higher stability against nucleases.
One skilled in the art would recognize that incorporating a palindrome into an immune stimulatory oligonucleotide could result in a molecule with undesired protein binding characteristics. For example, Liang et al., Mol. Cell. Biol. 16(7): 3773-3780 (1996) show that for GAL4, the palindromic CGG triplets at the ends of the 17-bp DNA recognition site are essential for tight binding and that PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp DNA recognition sequence. In addition, Nguyen et al., J Biol. Chem. 275:15466-15473 (2000) show that a palindromic sequence within the hNQO1 ARE/EpRE DNA recognition motif is necessary for binding of Nrf2/MafK heterodimer and cannot be competed by the ARE/EpRE sequence of rGSTA2, which does not contain the palindromic sequence. This binding of proteins to oligonucleotides is recognized as a limiting factor in using those oligonucleotides for immune stimulation compositions. However, incorporating a chemically modified palindrome into an immune modulatory oligonucleotide and/or 3′-3′ linking of chemically modified palindromic immune modulatory oligonucleotides could produce a molecule with the desired immune modulatory characteristics without the limitations of a natural, linear palindrome.
Thus, there remains a need to develop palindrome-containing compounds that modulate an immune response through TLRs but that are not plagued with the problems of linear oligonucleotides containing unmodified palindromic segments.